The presynaptic nerve terminal is a complex structure containing many proteins that control neurotransmitter release. Identifying the various regulatory proteins and elucidating their mechanisms of action are critical to understanding how the presynaptic terminal regulates neurotransmitter release rapidly and dynamically. The BK channel, a high-conductance and voltage/calcium-gated potassium channel, colocalizes with voltage-gated calcium channels at the presynaptic terminal and serves as a powerful negative regulator of neurotransmitter release. To identify novel regulators of neurotransmitter release, a genetic screen was performed to isolate mutants suppressing a lethargic phenotype caused by a hyperactive BK channel in C. elegans. Mutants of bkip-2 and bkip-4 (bkip for BK channel Interacting Protein) were isolated. BKIP-2 and BKIP-4 both have mammalian homologues with unknown functions in the brain. Preliminary studies suggest that BKIP-2 is required for BK channel function in neurons whereas BKIP-4 regulates neurotransmitter release through a mechanism that appears to be independent of the BK channel. The specific aims of this proposal are: (1) test the hypothesis that BKIP-4 regulates neurotransmitter release through controlling presynaptic Ca2+ concentration; (2) determine whether BKIP- 4 mutation alters neural anatomy, and whether BKIP-4 function is conserved in mammals; and (3) determine how BKIP-2 regulates the function of presynaptic BK channels. The long-term goals are to answer the important questions how various presynaptic proteins interact to shape neural circuits dynamically and why mutations of these proteins may cause diverse human diseases.